Usenobong F. Ufot^1,, Aniefiok E. Ite^{2, 3}, Idorenyin H. Usoh¹, Monday I. Akpanabiatu⁴

This study investigated the steady-state kinetics of engineered wild-type and manganese (II) binding site mutants of recombinant Phlebia radiata manganese peroxidase 3(rPr-MnP3). The effect (activation or inhibition) of some metal ions (Co²⁺, Zn²⁺ Cu²⁺ and Na⁺) on the activity of rPr-MnP3 enzymes was also studied. The results obtained showed that the rPr-MnP3 mutants in which the metal binding functionality has been largely lost have been created. Na⁺ (mono-valent ion) and Co²⁺showed similar characteristics by exhibiting stimulatory effects on the activity of wild-type rPr-MnP3. However, Cu²⁺ and Zn²⁺ had mixed inhibitory effects on wild-type and mutants (E40H, E44H, E40H/E44H). It was observed that Cu²⁺ was by far the strongest inhibitor of engineered rPr-MnP3 enzymes while Co²⁺ exhibited a non-competitive inhibitory effect on the double mutant (E40H/E44H) and D186H activities. In addition, Zn²⁺ and Cu²⁺also had non-competitive inhibitory effect on D186H mutant enzyme activity. The results obtained further showed that the competitive inhibitory effect of Cu²⁺observed in other rPr-MnP3 enzymes is largely removed in D186H mutant enzyme. Generally, histidine substitution retained a strong selectivity for Cu²⁺ as competitive inhibitor. Zn²⁺ being generally non-competitive suggest involvement of sites other than the Mn (II) binding site. This study showed that rPr-MnP3 enzymes function with alternate ligands in the Mn²⁺ binding site and does not have absolute obligate requirement for all carboxylate ligand set.

peroxidase, Phlebiaradiata, steady-state, wild-type, mutants, metal ions, inhibitors